Composition for repression of hyperlipidemia and obesity through suppression of intestinal cholesterol absorption

ABSTRACT

Disclosed are a composition for inhibiting hyperlipidemia and obesity through suppression of intestinal cholesterol absorption. An IgY-type antibody derived from yolk to NPC1L1 (Niemann-Pick C1-Like1), contained, as an active ingredient, in the composition of the present invention is linked to NPC1L1 (Niemann-Pick C1-Like1) that is a cholesterol transport protein in the intestines, thus interfering with binding between cholesterol and the transport protein to completely block absorption of cholesterol in the body and thereby prevent hyperlipidemia and obesity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase, pursuant to 35 U.S.C. §371,of PCT international application Ser. No. PCT/KR2011/003007, filed Apr.25, 2011, designation the United States and published in Korean on Dec.15, 2011 as publication WO2011/155705. PCT/KR2011/003007 claims priorityto Korean Patent Application Ser. 10-2010-0055000, filed Jun. 10, 2010.The entire contents of the aforementioned patent application areincorporated herein by reference.

SENQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 17, 2012, isnamed 912850_(—)306147. txt and is 29,521 bytes in size.

TECHNICAL FIELD

The present invention relates to a composition for inhibitinghyperlipidemia and obesity. More specifically, the present relates to acomposition for inhibiting hyperlipidemia and obesity throughsuppression of intestinal cholesterol absorption.

BACKGROUND ART

Cholesterol is known to be a factor that induces coronary cardiovasculardiseases which are known to be 30% or more of all causes of death atpresent.

Cardiovascular diseases are considered to be diseases of developingcountries having high fat intake and high obesity population and theonset rate thereof in Korea is rapidly increasing due to westernizationof diet, lack of exercise, overwork and the like in accordance withdevelopment of economical level. In particular, low-density lipoprotein(LDL) that is involved in transport of cholesterol in blood is regardedas a specific arteriosclerosis-inducing factor and oxidized LDL is knownto exhibit potent arteriosclerosis induction activity.

Meanwhile, obesity is commonly referred to as a phenomenon in whichresidual calories left after indigestion and consumption are convertedinto adipocyte and are deposited in various body sites, in particular,subcutaneous tissues and the abdominal cavity. The causes of obesityinclude genetic factors, environmental factors, energy metabolicdisorders and the like. The types of obesity may be classified intosimple (primary) obesity and symptomatic (secondary) obesity dependingon the onset cause thereof.

The most of obesity patients suffers from simple (primary) obesity andis known to be caused by accumulation of surplus energy in fat due tocalorie over-uptake and consumption lack of calorie in the body thereof.

Symptomatic (secondary) obesity is known to result from diseases such ashypothyroidism, adrenocortical hormone over-secretion and polycysticovary syndrome, or drugs such as oral contraceptives, tranquilizers,steroid hormones, drugs containing an antihistamine ingredient and thelike.

Obesity causes constipation, dyspepsia, gastroenteric troubles due toabdominal press by fat tissues, induces adult diseases such as diabetes,hypertension, arteriosclerosis, cardiac diseases and cancers, andcomplications thereof as well as mental diseases such as dissatisfactionassociated with the body, anxiety, personality disorders and depression.That is, obesity is a cause of all kinds of diseases.

Accordingly, it is necessary to reduce the amount of cholesterolabsorbed in the body and thereby prevent cardiovascular diseases andobesity that is a cause of all kinds of diseases. For this purpose,blocking of cholesterol absorption is the most efficient method.

Meanwhile, Ezetimibe is known in the art to be a compound that inhibitsabsorption of cholesterol, which is also called “Zetia”. Taking intoconsideration the market associated with obesity that graduallyincreases in demand, development of novel alternative substances isrequired.

DISCLOSURE Technical Problem

Therefore, the present invention has been made in view of the aboveproblems, and it is one object of the present invention to develop andprovide a composition that is capable of inhibiting or preventinghyperlipidemia and obesity by reducing the amount of cholesterol that isabsorbed in the intestines.

Technical Solution

In accordance with one aspect of the present invention, provided is acomposition for inhibiting absorption of cholesterol comprising, as anactive ingredient, an IgY-type antibody against an antigen thatcomprises, as an epitope, the entirety or part of amino acid sequencesin the loop part that protrudes towards the lumen among NPC1L1(Niemann-Pick C1-Like1) that is an intestinal cholesterol transportprotein.

In accordance with another aspect of the present invention, provided isa composition for preventing or inhibiting obesity comprising, as anactive ingredient, an IgY-type antibody against an antigen thatcomprises, as an epitope, the entirety or part of amino acid sequencesin the loop part that protrudes towards the lumen among NPC1L1(Niemann-Pick C1-Like1) that is an intestinal cholesterol transportprotein.

In accordance with yet another aspect of the present invention, providedis a composition for preventing or inhibiting hyperlipidemia comprising,as an active ingredient, an IgY-type antibody against an antigen thatcomprises, as an epitope, the entirety or part of amino acid sequencesin the loop part that protrudes towards the lumen among NPC1L1(Niemann-Pick C1-Like1) that is an intestinal cholesterol transportprotein.

Hereinafter, the present invention will be described in more detail.

The first, second and third aspects of the present invention provide thecomposition for inhibiting absorption of cholesterol, the compositionfor preventing or inhibiting obesity and the composition for preventingor inhibiting hyperlipidemia. All three aspects comprise, as an activeingredient, an IgY-type antibody against an antigen that comprises, asan epitope, the entirety or part of amino acid sequences in the looppart that protrudes towards the lumen among NPC1L1 (Niemann-PickC1-Like1) that is an intestinal cholesterol transport protein.

As can be seen from the following tests of the present invention, theIgY-type antibody against an antigen that comprises, as an epitope, theentirety or part of amino acid sequences in the loop part that protrudestowards the lumen among NPC1L1 (Niemann-Pick C1-Like1) that is anintestinal cholesterol transport protein effectively inhibits absorptionof cholesterol in the intestine (see FIG. 1 for inhibition mechanism).

Based on the aforementioned effects of absorption inhibition ofcholesterol, the IgY-type antibody against an antigen that comprises, asan epitope, the entirety or part of amino acid sequences in the looppart that protrudes towards the lumen among NPC1L1 (Niemann-PickC1-Like1) that is an intestinal cholesterol transport protein can beprepared and used as a composition for inhibiting absorption ofcholesterol and a composition for preventing or inhibiting obesity andhyperlipidemia that result from overtake of cholesterol.

Meanwhile, NPC1L1 (Niemann-Pick C1-Like1) protein used in the presentinvention is a cholesterol transport protein present in the intestine.For example, human contains a nucleic acid sequence represented bysequence number 1 and an amino acid sequence represented by a sequencenumber 2. NPC1L1 is known to have an important role in absorption ofcholesterol in small intestine. The absorption of cholesterol by NPC1L1protein through endocytic recycling contributes to unidirectional (invivo) absorption and is not affected by HDL, intracellular cholesterolabsorption and concentration in blood. Also, NPC1L1 is known toselectively recognize non-esterified free cholesterol and thusfacilitate unidirectional transport into hepatoma cells (J. Mark Brownat. al., Biochem. J. (2007) 406, 273-283).

In the present invention, an antibody to block the action of NPC1L1described above is prepared, and in particular, an IgY-type antibodyagainst an antigen that comprises, as an epitope, the entirety or partof amino acid sequences in the loop part that protrudes towards thelumen among NPC1L1 (Niemann-Pick C1-Like1) is produced and used. Sincethe sequence of the loop part that protrudes towards the lumen amongNPC1L1 (Niemann-Pick C1-Like1) is known, the loop part is produced intopeptide through biosynthesis or genetic recombination and may be used asan epitope.

Meanwhile, IgY is an antibody contained in yolk and an antibodydeveloped into an IgY-type is known to have almost no side effects wheningested in the human body. IgY may be produced by injecting an antigeninto a chicken. This method is known in the art and a detailedexplanation thereof is thus omitted.

Meanwhile, the term “active ingredient” used herein means that theinhibitory effect of absorption of cholesterol in the composition andinhibitory effect of hyperlipidemia or obesity are derived from“IgY-type antibody” provided in the present invention, and means that avariety of aid ingredients other than these ingredients may be added inorder to facilitate preservability and absorption.

Meanwhile, the loops that protrude towards the lumen among NPC1L1(Niemann-Pick C1-Like1) are seven in total, have amino acid sequencesrepresented by sequence numbers 4, 6, 8, 10, 12, 14 and 16, and are usedas epitopes for production of antibodies. Accordingly, the amino acidsequence present in the loop part that protrudes towards the lumen amongNPC1L1 that can be used as epitopes in the present invention is one ofamino acid sequences represented by sequence numbers 4, 6, 8, 10, 12, 14and 16.

Meanwhile, in the present invention, the antigen used for production ofIgY-type anti-body may be formed by binding a carrier protein that caninduce antigenicity to the entirety or part of amino acid sequences inthe loop part that protrudes towards the lumen among NPC1L1(Niemann-Pick C1-Like1). As molecular weight increases, antigenicity canbe improved. At this time, as the carrier protein that inducesantigenicity, one selected from bovine serum albumin (BSA), keyholelimpet haemocyanine (KLH) and ovalbumin (OVA) may be used.

Adavantagous Effects

The IgY-type antibody derived from yolk to NPC1L1 (Niemann-PickC1-Like1), contained, as an active ingredient, in the composition of thepresent invention is linked to NPC1L1 (Niemann-Pick C1-Like1) that is acholesterol transport protein in the intestines, thus interfering withbinding between cholesterol and the transport protein to completelyblock absorption of cholesterol in the body and thereby preventhyperlipidemia and obesity.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 is a schematic view illustrating a mechanism in which ananti-NPC1L1 IgY inhibits absorption of cholesterol;

FIG. 2 illustrates test results of production of recombinant antigenthat produces IgY and antigenicity of the produced antigen;

FIG. 3 illustrates electrophoresis results of the produced recombinantan antibody, IgY;

FIG. 4 illustrates western blot results of the binding capability of arecombinant antibody, IgY to antigen;

FIG. 5 illustrates ELISA results quantitatively showing whether or notvaccine forms an antibody, IgY;

FIG. 6 illustrates in vitro immunofluorescence results confirmingbinding of IgY to NPC1L1 protein using HepG2 cell lines. The results areobtained by observation at a magnification of 400× using a confocalmicroscope;

FIG. 7 illustrates in vivo immunohistochemistry results confirmingbonding of IgY to a NPC1L1 protein in the mouse small intestine tissue.The results are obtained by observation at a magnification of 200× usinga confocal microscope;

FIG. 8 illustrates in vivo immunofluorescence results confirming bindingof IgY to a NPC1L1 protein in mouse small intestine tissues. The resultsare obtained by observation at a magnification of 200× using a confocalmicroscope;

FIG. 9 illustrates cholesterol uptake inhibition test results usingHepG2 cell lines. In FIG. 9, ‘COM’ and ‘ISA’ mean the kinds of adjuvantand ISA means an anti-NPC1L1 IgY sample produced using ‘ISA 70adjuvant’, and ‘COM’ means an anti-NPC1L1 IgY sample produced using a‘Complete Freund adjuvant (Difco, USA)’; and

FIG. 10 illustrates variation in body weight while a high cholesterolfeed was ingested over 8 weeks (*; p<0.05, **; p<0.01).

BEST MODE

Hereinafter, the following examples will be provided for a furtherunderstanding of the invention. The scope of the present invention isnot limited to the following examples and includes technical spiritsequivalent thereto.

PRODUCTION EXAMPLE 1 Production of Recombinant Antigen and Confirmationof Suitability

Hereinafter, a recombinant antigen (hereinafter, referred to as ‘protein373’) was produced by cloning a total of 262 amino acids represented byamino acid sequence numbers 373 to 634 so that the recombinant antigenhas amino acid of loop 1 (the second loop, among a total of seven loopsthat NPC1L1 have in a lumen direction) represented by sequence number 4,among the overall amino acid sequence of NPC1L1.

Also, a recombinant antigen (hereinafter, referred to as ‘protein 416’)was produced by cloning a total of 220 amino acids represented by aminoacid sequence numbers 416 to 635 among the overall amino acid sequenceof NPC1L1.

Also, a recombinant antigen (hereinafter, referred to as ‘protein 509’)was produced by cloning a total of 125 amino acids represented by aminoacid sequence numbers 509 to 633 among the overall amino acid sequenceof NPC1L1.

The respective cloned DNA sequences used for production of antigens toproduce IgY were ligated into the Xho I/BamH I cloning site of pET-15bvector having a His-tag for purification and then expressed in an E.coli BL21 (DE3) host using IPTG.

After over-expression, the DNA sequences were purified on a His6affinity column and solubilized, and the resulting recombinant proteinwas subjected to SDS-PAGE and western blotting.

The antibodies used for western blotting herein were commerciallyavailable anti-NPC1L1 mouse monoclonal antibodies and HRP-conjugatedanti-mouse goat antibodies.

The test results are shown in FIG. 2. From the fact that theover-expressed and purified recombinant NPC1L1 antigens (proteins 373,416 and 509) reacted with anti-NPC1L1 antibodies derived from the mouse,it could be seen that the NPC1L1 antigens were produced as recombinantproteins having a main epitope and had an antibody property of producingIgY that inhibits the function of NPC1L1 protein.

EXAMPLE Production of IgY Antibody Through Vaccine of RecombinantAntigen

(1) Preparation of vaccine

A vaccine was prepared by mixing the recombinant peptide-typeantibody-type NPC1L1 antigen produced in Production Example 1 above andFreund's complete adjuvant (Difco 263810, USA) at equivalent volumes. Inorder to confirm difference in formation of antibodies depending onpresence of adjuvant, ISA70 (general adjuvant) and an antigen were mixedat equivalent amounts using a syringe to prepare a vaccine forproduction of specific-yolk antibody.

The conjugation between the recombinant peptide and the carrier wasperformed using meleimide-activated BSA and KLH conjugated kit(Maleimide Activated BSA, KLH conjugation Kit, Sigma-Aldrich, MBK1, USA)and this method was performed in accordance with the instruction manual.The method will be described in brief. A carrier protein was dissolvedin pH 6.6 in 20 mM sodium phosphate, 230 mM NaCl, 2 mM EDTA, and 80 mMsucrose, recombinant peptide was dissolved in pH 6.6 in 20 mM sodiumphosphate, 100 mM EDTA and 80 mM EDTA, the carrier protein and therecombinant peptide were mixed and stirred in a refrigeration for 12hours or longer, and finally separated on a Sepadex G-25M gel filtrationcolumn.

(2) Immunization of Egg-Laying Chickens

The prepared vaccine was intramuscularally injected into 22-week oldHy-line brown egg-laying chickens, was primarily inoculated at aninterval of three weeks and was boosted twice.

(3) Separation and Confirmation of Immune-Yolk Antibody

i) Separation of Immune-Yolk Antibody

IgY was isolated from eggs produced from the immunized egg-layingchicken using ammonium sulfate (sigma USA). The ammonium sulfate methodwas carried out by removing an egg membrane from yolk, diluting with pH2.5 D.W at a ratio of 1:4, freezing the dilute at −20° C. for 2 days,centrifuging the dilute at 7000 rpm for 30 minutes, and filtering thesupernatant to separate a water soluble protein in accordance with themethod of Akita et., Al. (Akita, E. M. and Nakai, S. Immunoglobulinsfrom egg yolk: isolation and purification. J. Food. Sci., 57: 629-633,1992). A pure protein was precipitated from the separated protein withan over-saturated ammonium sulfate solution at 4° C. overnight. Theprecipitated solution was centrifuged to obtain a pellet, re-suspendedwith PBS, and dialyzed at 4° C. with a PBS buffer to harvest theseparated antibody sample.

ii) Electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) wasperformed using 5% stacking gel and 10% seperating gel) in accordancewith the method of Laemmli (Laemmli. U.K. Cleavage of structuralproteins during the assembly of the head of bacteriophage T4. Nature,227(5259): 680-685, 1970). After electrophoresis, the gel was stainedwith a Coomassie brilliant blue R-250 solution for 30 minutes and theseparated IgY antibody was confirmed using a destaining buffer. FIG. 3shows electrophoresis results of the produced recombinant antibody, IgY.

(4) Confirmation of Binding of Separated IgY to Antigen

The binding between the separated IgY antibody and peptide antigen wasconfirmed by western blotting. As a result, as shown in FIG. 4, it wasconfirmed that IgY produced by immunization recognized recombinantprotein that contained the C loop part of NPC1L1 and was conjugated.

Test Example 1 Confirmation of Formation of Antibody Using ELISA TestMethod

In this Test Example, whether or not an antibody was formed by thevaccine (protein 416) prepared using the complete adjuvant and the ISA70 adjuvant was confirmed in accordance with an ELISA test method.

a) The NPC1L1-BSA-conjugated antigen was coated at a concentration of400 ng/ml on a 96-well ELISA plate using a carbonate buffer andincubated at 37° C. for one hour to complete coating.

b) The antigen was washed with PBST three times and blocked with 1%-BSAat 37° C. for one hour.

c) The antigen was washed with PBST three times, was treated with 100 ulof a sample and incubated at 37□ for one hour.

d) The antigen was washed with PBST three times, treated with 100 ul ofanti-chicken-IgY-HRP as a secondary antibody and incubated at 37° C. forone hour.

e) The antigen was washed with PBST three times, 100 ul of a preparedsubstrate solution was added thereto, and color reaction was performedfor 10 minutes and was ceased with 2N sulfuric acid.

f) The results were confirmed using an ELISA reader.

In accordance with the test results (FIG. 5) and the results of antibodysamples diluted at a ratio of 1:10,000, when comparing the blank valuewith ISA70-IgY, and COM-IgY, the difference in O.D of about 10- to15-fold was observed. There was no difference in O.D between the sampleof the general IgY and the blank. This means that an anti-NPC1L1-IgYantibody was formed.

Also, regarding formation of antibody caused by the difference betweenadjuvants, the Freund's complete adjuvant (Difco 263810, USA) containingOMP of microorganisms exhibited superior titer, as compared to the ISA70 adjuvant.

From results of western blotting test of Example 1 and the present ELISAtest, it could be seen that the anti-NPC1L1-IgY antibody was well formedand an antibody was bound to the antigen.

Test Example 2 Immunofluorescence of In Vitro NPC1L1

In order to confirm whether the anti-NPC1L1 IgY antibody (antibody toprotein 416) separated from yolk was bound to an NPC1L1 protein as anantigen, hepatoma cell lines, HepG2 cell lines that are known toover-express NPC1L1 were subjected to in vitro immunofluorescence.(Davies J P, Scott C, Oishi K, Liapis A, Ioannou Y A. Inactivation ofNPC1L1 causes multiple lipid transport defects and protects againstdiet-induced hypercholesterolemia. J Biol. Chem. 2005 Apr. 1;280(13):12710-20. Epub 2005 Jan. 25.)

The cells were seeded at a concentration of 1×10⁴/ml on a slide chamber,incubated for 18 hours and then tested. The cell medium was removed, thecells were fixed with 3.7% formaldehyde, washed with PBST, treated witha permeabilization buffer (0.2% Triton X-100) for 20 minutes, each of2.5 ug/ml of anti-NPC1L1-IgY as a primary antibody andrabbit-anti-NPC1L1 (Santa Cruz, USA) as a commercial antibody wasdiluted at a ratio of 1/50 and the cells were treated with each dilutedantibody. The cells were washed with PBS, each of anti-chickenIgY-Alexa488 (Biotium, USA) as a secondary antibody and anti-rabbitIgG-Alexa488 (Invitrogen, USA) was diluted at a ratio of 1/100, thecells were treated with each diluted antibody at room temperature (RT)for one hour and washed with PBS, the nucleus was counterstained withHoechst33258 for 30 minutes and mounted, and the results were observedusing a multi-photon confocal laser scanning microscope (LSM 510 METANLO, Carl Zeiss, Germany).

As could be seen from the results of immunofluorescence (IF) (FIG. 6),except that a negative control that was not treated with a primaryantibody, the anti-NPC1L1 IgY antibody was bound to NPC1L1 as an antigenand expressed in cytoplasm, and the antibody produced from yolk was wellbound to the target protein.

Test Example 3 In Vivo Immunohistochemistry and Immunofluorescence

It could be seen from the results that the anti-NPC1L1-IgY (antibody toprotein 416) was in vitro bound to the NPC1L1 protein and,immunohistochemistry (IHC) and immunofluorescence (IF) were performedusing mouse intestine tissues in order to confirm whether or not theanti-NPC1L1-IgY was bound to the NPC1L1 protein actually present insmall intestines.

(1) In Vivo Immunohistochemistry

In order to confirm whether or not the anti-NPC1L1-IgY separated fromyolk was bound to the NPC1L1 protein, immunohistochemistry was performedon the mouse intestine tissues.

Altmann et., al. (Altmann S W, Davis H R Jr, Zhu L J, Yao X, Hoos L M,Tetzloff G, Iyer S P, Maguire M, Golovko A, Zeng M, Wang L, Murgolo N,Graziano M P. Niemann-Pick C1 Like 1 protein is critical for intestinalcholesterol absorption. Science. 2004 Feb. 20; 303(5661):1201-4)reported that a great amount of small intestinal NPC1L1s was distributedin the small intestine proximal part.

Accordingly, the mouse small intestine was harvested, the jejunum of theproximal part except the duodenum was separated and washed with PBS toremove foods present in the intestinal canal, fixed with 4%paraformaldehyde, fixed with paraffin using an automatic tissueprocessor (Leica, Germany), embedded (Leica, Germany) to produce aparaffin block, and cut to a size of 5 μm with a tissue microtome(Leica, Germany) to produce a slide sample for immunostaining.

The slide was deparaffinized with xylene, hydrated in ethanol series(100%, 95%, 90%, 80%, 70%, 50%), washed with PBS, treated with 0.3% H₂O₂to remove endogenous peroxidase, blocked with 5% normal serum (Vector,USA), treated with a predetermined concentration of anti-NPC1L1-IgY (2.5ug/ml) separated from yolk and a commercial antibody(rabbit-anti-NPC1L1, 1/50, Santa Cruz, USA) as primary antibodies, andincubated at 4° C. overnight. The sample was washed with PBS, treatedwith a diluted (1/100) secondary antibody (Anti-Chicken Biotin,Anti-Rabbit-Biotin, Vetor, USA) at room temperature (RT) for 2 hours,washed with PBS, and ABC was incubated using a VECTASTAIN ABC Kit(Vetor, USA) for 2 hours. The ABC was reacted with a DAB solution(Vetor, USA) for 2 to 5 minutes, washed with PBS, counter-stained withhematoxylin (Vector, USA), washed with D.W, dehydrated with ethanolseries, treated with xylene and mounted, and the results were observedusing an optical microscope (Carl Zeiss, Germany).

(2) In Vivo Immunofluorescence

In order to confirm whether or not the anti-NPC1L1-IgY separated fromyolk was bound to the NPC1L1 protein, immunofluorescence was performedon the mouse intestine tissues. The tissue paraffin xylene wasdeparaffinized with xylene, hydrated in ethanol series (100%, 95%, 90%,80%, 70%, 50%), washed with PBS, treated with 0.3% H₂O₂ to removeendogenous peroxidase, blocked with 5% normal serum (Vector, USA),treated with a predetermined concentration of anti-NPC1L1-IgY (2.5ug/ml) separated from yolk and a commercial antibody(rabbit-anti-NPC1L1, 1/50, Santa Cruz, USA) as primary antibodies, andincubated at 4° C. overnight. The sample was washed with PBS, treatedwith each 1/100 dilution of anti-chicken IgY-Alexa488 (Biotium, USA) andanti-rabbit IgG-Alexa488 (Invitrogen, USA) as secondary antibodies atroom temperature for 2 hours, washed with PBS, nucleus wascounterstained with Hoechst33258 and mounted, and the results wereobserved with an optical microscope (Carl Zeiss, Germany).

(3) Test Results

As can be seen from the results shown in FIGS. 7 and 8, the anti-NPC1L1IgY antibody was strongly expressed throughout the ileal villus distalpart and was expressed in the proximal part other than the villus distalpart, and, it was confirmed through IF that fluorescence was stronglyemitted in the form of a line in epithelial cells of the villus distalpart.

It could be seen from these results that IgY produced in the presentinvention was actually bound even in small intestines.

Test Example 4 Cholesterol Uptake Assay of Anti-NPC1L1 IgY antibody

In order to confirm efficacies and effects of anti-NPC1L1 IgY (antibodyto protein 416) cholesterol uptake assay was performed using Hep G2cells.

Hep G2 cells were incubated at a density of 2×10⁵/ml on a 24-well plateusing a DMEM (Difco, USA) medium containing 10% FBS (Difco, USA) for 18hours, treated with anti-NPC1L1 IgY at different concentrations (5, 25and 50 ug/ml), and treated with 10 ug/ml of Ezetimibe known as acholesterol uptake inhibitor as a positive control. Each sample waspre-incubated at 37° C. for one hour and then was removed, and a freshmedium was added thereto, followed by washing. The sample was treatedwith 50 uM of radio isotope-labeled [3H]-cholesterol for 3 hours. Thecells were washed with 0.1% fatty acid-free BAS-containing PBS,collected with HESS (Difco, USA), cytolysis was performed with 1% TritonX-100 (Sigma, USA)-containing HESS, radiation dose was measured with aBeckmen LS6500 scintillation counter, and comparison and assay wereperformed using a group to which IgY was administered as a controlgroup.

As can be seen from the result of FIG. 9, as compared to the non-treatedcontrol group, Ezetimibe serving as a positive control exhibited asignificant decrease (p<0.05) at a concentration of 10 ug/ml, and IgYexhibited a significant decrease (p<0.05) in cholesterol uptake at aconcentration of 25 ug/ml. In FIG. 9, ‘COM’ and ‘ISA’ represent thetypes of adjuvant, “ISA” means an anti-NPC1L1 IgY treatment group using‘ISA 70 adjuvant’, and ‘COM’ means an anti-NPC1L1 IgY treatment groupusing a ‘Complete Freund adjuvant (Difco, USA)’.

These results indicate that the produced anti-NPC1L1 IgY antibody iseffectively bound to the NPC1L1 protein, and the bound IgY significantlyinhibits cholesterol uptake of NPC1L1. That is, the anti-NPC1L1 IgY ofthe present invention is an antibody that has the same effects asEzetimibe, the drug, known to be a conventional cholesterol uptakeinhibitor.

Test Example 5 Animal Test of Anti-NPC1L1 Igy Antibody

An animal test was performed in order to confirm efficacies ofanti-NPC1L1 IgY antibody (antibody to protein 416) bound to NPC1L1(Niemann-Pick C1 Like1), intestinal cholesterol transport protein.

(1) Test Animal

The test animals used herein were 5-week old C57BL/6 female miceavailable from KOATECH (Korea) and the animals were acclimatized for 7days, animals were classified into groups (n=10) the day before of thetest, and grouping was performed in a state that the average weight ofall test groups was identical. The test animals were grown in apolycarbonate cage (width 26 cm, length 42 cm, height 18 cm), and werebred while they were fed with sterilized distilled water and feed withexperimental animal (Purina Korea, Inc.), all groups except the normalgroup were freely fed with an atherogenic diet (available from CentralLab. Animal Inc. D12336, Research diets, INC. USA) so that they digesteda high concentration of cholesterol during the test term. The testanimals were tested in accordance with the instructions of test animalethics commission in Chuncheon bioindustry foundation.

(2) Administration of Test Group and Test Material

The average weight of all test groups was made identical and test groupswere classified. The animals were classified into five groups in total,and were roughly divided into a normal group that was not treated and agroup to which a high cholesterol feed was administered, a control groupfor high cholesterol feed, and an IgY control group and an IgYadministration group that were classified depending on theadministration of IgY. IgY was administered in amounts of 50 mg and 250mg per animal weight kg, anti-helicobacter pylori IgY was administeredin an amount of 250 mg per animal weight kg to an IgY control group, andPBS was administered to the normal group and the control group.

(3) Measurement of Body Weight

The test animals were labeled on the ear thereof using an ear punch foranimals in accordance with an individual identification method, and aweight increase percentage was measured with respect to 100% of the testinitial weight. The weight was measured at a predetermined time once aweek.

(4) Statistical Treatment

The significance of the test results was identified by a one wayANOVA-test using a GraphPad 4.0 prism program and was expressed withrespect to the control group to which the high cholesterol feed wasadministered.

(5) Test Results

As can be seen from FIG. 10, as a result of administration of highcholesterol feed, the weight increase percentage was rapidly elevated atthree weeks after the test, and a weight increase percentage showing abroad curve was observed after 6 weeks, and assuming that the testinitial weight was 100%, the control group to which a high cholesterolfeed was administered exhibited a 41% increase in weight, while thegroup to which the anti-NPC1L1-IgY was administered exhibited 30% and29% increase percentages in weight, which means that the increasepercentage in body weight caused by the feed was significantly decreased(p<0.01).

Also, in order to confirm the effects of administration of IgY, thegroup to which the anti-America helicobacter pylori IgY was administereddid not exhibit the inhibitory effect of administered IgY on the bodyweight increase.

Accordingly, it may be thought that IgY effectively acts on intestinalcholesterol transport protein, NPC1L1, thus significantly inhibiting aweight increase.

Although the preferred embodiments of the present invention have beendisclosed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

[Sequence Free Text]

Seq. No. 1 represents a nucleic acid sequence that encodes NPC1L1(Niemann-Pick C1 Like 1), cholesterol transport proteins present in theintestines of the human.

Seq. No. 2 represents an amino acid sequence that encodes NPC1L1(Niemann-Pick C1 Like 1), cholesterol transport proteins present in theintestines of the human.

Seq. Nos. 4, 6, 8, 10, 12, 14 and 16 represent amino acid sequences ofseven loops that protrude towards the lumen among NPC1L1 (Niemann-PickC1-Like 1).

Seq. Nos. 3, 5, 7, 9, 11, 13 and 15 represent nucleic acid sequencesthat encode Seq. Nos. 4, 6, 8, 10, 12, 14 and 16 amino acid sequences,respectively.

The invention claimed is:
 1. A food composition for treating obesitycomprising, as an active ingredient, an IgY-type antibody against anantigen that comprises, as an epitope, amino acid sequence numbers 416to 635 of NPC1L1 (Niemann-Pick C1-Like1) that is an intestinalcholesterol transport protein encoded by the amino acid sequence of SEQ.ID. No:2.
 2. A food composition for treating hyperlipidemia comprising,as an active ingredient, an IgY-type antibody against an antigen thatcomprises, as an epitope, amino acid sequence numbers 416 to 635 ofNPC1L1 (Niemann-Pick C1-Like1) that is an intestinal cholesteroltransport protein encoded by the amino acid sequence of SEQ. ID. No:2.